A novel protein engineering tool for rapid manufacturing of designer nucleosomes

PROJECT SUMMARY Nucleosomes are the basic building blocks of chromatin and composed of bp DNA wrapped around a core histone octamer Chromatin function is controlled by the dynamic addition removal of histone post translational modifications PTMs Significantly alterations in specific PTMs are associated with changes in gene expression driving the emergence progression of many cancer pathologies Mounting evidence indicates that screening chromatin modifiers in the context of nucleosomal substrates is essential to recapitulate specificity toward appropriate PTM sites and thus biologically relevant activity However PTM regulators continue to be studied via modified histone fragments which poorly mimic chromatin regulation in vivo EpiCypher is pioneering the commercial development of nucleosomes carrying disease relevant PTMs `designer nucleosomesandapos or `dNucsandapos for innovative drug discovery development The current leading technology to generate modified histones for dNuc assembly is native chemical ligation NCL which permits the scarless incorporation of diverse PTMs e g methylation acetylation and phosphorylation on a single histone subunit However NCL is a multistep and labor intensive process that requires weeks of dedicated effort to synthesize a single dNuc in the milligram scale Therefore a new methodology is greatly needed to support the rapid generation of dNucs with diverse PTM layouts To meet this need EpiCypher is joining with Dr Matthew Levy from The Albert Einstein College of Medicine to develop a novel protein engineering tool for accelerated dNuc manufacturing The Staphylococcus aureus Sortase A SrtA transpeptidase can be modified by directed evolution to alter its recognition sequence LPXTG where X any amino acid to seamlessly ligate `unnaturalandapos protein substrates Dr Levyandapos s group recently developed a powerful directed evolution approach to rapidly screen large mutant libraries generating a robust SrtA SrtA variant with improved activity over wild type enzyme In Aim Dr Levyandapos s team will further evolve this highly active SrtA variant toward histone H to rapidly incorporate PTMs into this substrate In Aim EpiCypher will demonstrate how this tool can be used for accelerated dNuc manufacturing First we will optimize a single step ligation reaction to synthesize large quantities of ultrapure dNucs for high throughput assay development We will also optimize ligation of modified peptides directly to pre assembled nucleosomes providing a powerful diversity manufacturing platform capable of multiplex synthesis In Phase we will utilize the SrtA H variant developed here to scale up manufacturing of a diverse catalog of dNucs with disease associated PTMs In addition we will optimize multiplexed dNuc manufacturing to develop dNuc based arrays for drug discovery Finally we will continue to generate new SrtA variants that selectively target histone H The breakout technology described herein will provide transformational opportunities for the development of next generation cancer therapeutics and epigenetic tool development PROJECT NARRATIVE Semi synthetic nucleosomes carrying specific post translational modifications known as designer nucleosomes represent powerful substrates for novel drug discovery and development However their potential is limited by the large investment of time and resources currently required for their synthesis Here EpiCypher outline plans to vastly increase dNUC manufacturing throughput The innovative technology we propose will enable the rapid generation of a diverse catalog of nucleosomes carrying disease relevant modifications for high throughput drug screening applications or novel tool development